Taxane Resistance in Breast and Ovarian Cancer Cells

描述（由申请人提供）：本提案将研究乳腺癌和卵巢癌紫杉烷治疗的非MDR 1遗传和细胞决定因素。我们已经从4个乳腺癌细胞系和6个卵巢癌细胞系中建立了17个紫杉烷耐药变异体，我们假设这些不同的变异体可能会深入了解紫杉烷耐药的新机制。具体目标是：1.确定微管蛋白异构体含量和微管动态不稳定性的潜在变化。我们对6类β-微管蛋白亚型表达的分析揭示了许多紫杉烷耐药变异体中β-微管蛋白含量的改变。将与玛丽安·乔丹博士合作评估这些细胞中的微管组装。2.探讨微管蛋白和微管相关蛋白基因改变对药物敏感性的影响。几个变体下调MAP 4和上调MAP Tau，我们假设这些改变可能通过改变紫杉烷结合对紫杉烷敏感性产生不利影响。我们计划使用siRNA技术来研究这些基因和特定微管蛋白亚型的作用，并且利用四环素调控的诱导型载体将用于将感兴趣的基因转染到亲本细胞中。3.探讨细胞凋亡（如Bcl-2、Bcl-XL、Akt）和细胞生长（如EGFR-1、her 2）的调节因子作为紫杉烷类药物应答的决定因素。几种非P-gp变体具有改变的凋亡调节因子表达。我们将使用反义和RNAi方法来研究抑制这些调节因子对细胞对紫杉烷敏感性的影响。我们还将通过使用激酶抑制剂抑制EGFR-1、her 2、Akt和其他基因来确定抑制生长途径对紫杉烷敏感性的影响。4.寻找新的紫杉烷类药物耐药预测基因。遗传（阵列CGH）和基因组（微阵列基因表达谱）的方法将被用来寻找新的遗传标记的预测敏感性和耐药性紫杉烷治疗。候选基因将通过PCR、免疫印迹和tet调节的表达使用我们已经制备的MCF-7 Flp-ln系进行验证。5.研究乳腺癌和卵巢癌临床标本对紫杉烷类药物的耐药性。将对来自NCI Cooperative Human Tissue Network的乳腺癌和卵巢癌肿瘤标本进行全基因组分析，并注释对紫杉烷治疗的反应性。还将在这些标本中验证细胞模型中鉴定的耐药因子表达。总之，该项目将使用大量的卵巢癌和乳腺癌细胞系中对紫杉烷类的细胞耐药的独特模型以及从接受紫杉烷类治疗的患者中获得的临床标本：（1）研究已知耐药机制与这些药物的相关性，（2）探索新的机制，（3）开发临床反应标志物。

DESCRIPTION (provided by applicant): This proposal will study non-MDR1 genetic and cellular determinants of taxane therapy in breast and ovarian cancers. We have established 17 taxane-resistant variants from 4 breast and 6 ovarian cancer cell lines, and we hypothesize that these diverse variants may yield insight into novel mechanisms of resistance to taxanes. The specific aims are: 1. To determine tubulin isoform content and potential alterations in microtubule dynamic instability. Our analyses of the expression of the six classes of (3- tubulin isoforms reveal altered p-tubulin content in many of the taxane-resistant variants. Microtubule assembly in these cells will be assessed in collaboration with Dr. Mary Ann Jordan. 2. To study the effects of altered tubulin and microtubule associated protein genes on drug sensitivity. Several of the variants have down-regulated MAP4 and up-regulated MAP Tau, and we hypothesize that these alterations may have an adverse effect on taxane sensitivity by altering taxane binding. We plan to use siRNA technology to study the effects of these genes and specific tubulin isoforms, and inducible vectors utilizing tetracycline regulation will be used to transfect genes of interest into parental cells. 3. To explore regulators of apoptosis (e.g. Bcl-2, Bcl-XL, Akt) and cell growth (e.g. EGFR-1, her2) as determinants of response to taxanes. Several of the non-P-gp variants possess altered expression of apoptotic regulators. We will use antisense and RNAi approaches to study the effects of inhibition of these regulators on cellular sensitivity to taxanes. We will also determine the effects of inhibition of growth pathways on taxane sensitivity by inhibiting EGFR-1, her2, Akt and other genes using kinase inhibitors. 4. To identify novel genetic markers to predict resistance to taxanes. Genetic (array CGH) and genomic (microarray gene expression profiling) approaches will be used to search for novel genetic markers for the prediction of sensitivity and resistance to taxane therapy. Candidate genes will be validated by PCR, immunoblotting, and tet-regulated expression using an MCF-7 Flp-ln line which we have made. 5. To study taxane resistance in clinical specimens of breast and ovarian cancer. Whole genome profiling of tumor specimens from breast and ovarian cancers from the NCI Cooperative Human Tissue Network, annotated for responsiveness to taxane therapy, will be performed. Expression of resistance factors identified in cellular models will also be validated in these specimens. In summary, this project will use a large set of unique models of cellular resistance to taxanes in ovarian and breast cancer cell lines and clinical specimens obtained from patients treated with taxanes to: (1) study the relevance of known resistance mechanisms to these drugs, (2) explore new mechanisms, and (3) develop clinical markers for response.